International Journal of Agricultural

Science and Research (IJASR)

ISSN(P): 2250-0057; ISSN(E): 2321-0087
Vol. 7, Issue 1, Feb 2017, 327-334
TJPRC Pvt. Ltd.

ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF METHANOL

EXTRACT OF BLEPHARIS GLOMERANS FLOWERS

SIVA KESAVARAO KOMMULA1, PRASAD UNDRAJAVARAPU2 &

KRISHNA SOWJANYA KANCHANA3
1
Department of Biochemistry, Dr Y.S.R. Horticultural University, Anantharajupeta, Andhra Pradesh, India
2
Department of Biotechnology, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India
3
Department of Biotechnology, Andhra University, Visakhapatnam, Andhra Pradesh, India
ABSTRACT

This study was undertaken to investigate in-vitro antioxidant and antibacterial activities of Blepharis
glomerans flowers (BGF). In this experiment we used methanol for the soxhlet extraction. Methanol extract of BGF
comprises alkaloids, cardiac glycosides, steroids, saponins, flavonoids and terpenoids. Phenol content of the BGF was
51.4 mg/g and flavonoid content was 38.9 mg/g. Methanol extracts of BGF showed DPPH (47.7%) and ABTS (44.8%)
activity. Furthermore, the methanol extract was used in antibacterial studies, which shows the highest inhibitory activity
against K. pneumoniae and least inhibitory activity against S. typhi. This study explored BGF as a potential source of

Original Article
antioxidant as well as antibacterial properties.

KEYWORDS: Blepharis Glomerans, Antioxidant Activity & Antibacterial activity

Received: Dec 07, 2016; Accepted: Jan 16, 2017; Published: Jan 28, 2017; Paper Id.: IJASRFEB201743

INTRODUCTION

Vegetation reminiscent of herbs, shrubs and climbers are exploited for their bioactive compounds for
human wellness (Ezzatzadeh et al., 2012). In addition, plants contain major bioactive constituents such as
alkaloids, flavonoids, saponins, steroids, terpenoids, polysaccharides and tannins, which can be mostly
contributing to biochemical events and latest therapeutic principles (Doughari et al., 2012). Herbal food
supplements or alternative therapies or natural/plant based approach are being used to control various bacterial and
fungal diseases (Prasannabalaji et al., 2012). The interaction of different groups of active metabolites within the
extract could have enhanced the therapeutic outcomes more than the only components
(Prasannabalaji et al., 2010). Oxidative stress is caused by an imbalance between the production of reactive
oxygen and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting
damage. Biochemical pathways or cellular mechanisms had been producing free radicals and reactive oxygen
species as an end product (Nantitanon et al., 2007). Unstable chemicals are harmful to living cells, it can cause
mutation, myocardial infarction, Alzheimer diseases and can be related to different scientific problems
(Adiguzel et al., 2009; Sugumaran and Raj, 2010). Usually, chemical centered antioxidants are used to manipulate
free radical activity and they have opposed results on human health and the development of normal antioxidant
from plant assets could be fruitful to humankind (Rajasekar et al., 2011).

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328 Siva Kesavarao Kommula, Prasad Undrajavarapu & Krishna Sowjanya Kanchana

Blepharis the most important genus of Acanthaceae; contemporary monographs by way of Vollesen (2000) as
containing 129 species. Quite a lot of species of Blepharis have confirmed medicinal values like anti-inflammatory,
antiarthritic, antimicrobial, antifungal, antioxidant and cytotoxic properties. It was once pronounced that B.Edulis has been
proven as a strong aphrodisiac undertaking, antispasmodic and antiplatelet aggregation (Fatima S et al., 2012). B.Ciliaris
has shown antioxidant activity (Mohammed et al., 2012), and seeds have confirmed expectorant, aphrodisiac, and diuretic
(Deshpande et al., 2006). Therefore the genus Blepharis has been validated for its medicinal values in previous studies.

MATERIALS AND METHODS

Plant Collection and Identification

Blepharis glomerans flowers were collected from the koyathanda at srisailam region, Andhrapradesh, India.
The collected flowers were washed twice in running tap water to remove clay and grimes. Fresh flowers were lyophilized
and then pulverized into powder form and sieved with 50 m mesh. Methanol is used as a solvent in this experiment.

Hot Extraction Method by Soxhlet

20 g of plant powder was extracted in 500 ml of methanol (by soxhlet extraction technique overnight at 65C
temperatures). The acquired extracts have been subjected to vacuum evaporator to remove excess solvents. Then the dried
crude extract yield has been weighed and used for additional experimental studies.

Phytochemical Screening of Methanolic Extracts

The small amount of crude methanol extract of BGF was dissolved in a suitable solvent and applied as a small
spot on the activated thin layer chromatography (TLC) plate. The ensuing plate runs with solvent methods
(100% chloroform, 70% chloroform+30% n-hexane, 50% chloroform+50% n-hexane, 30% chloroform+30%
n-hexane+40% methanol) and visualized with quite a lot of spray reagents (vanillin-sulfuric acid spray, sulfate-sulfuric
acid spray, Dragendorffs spray, aluminium chloride spray, 4-aminoantipyrine/potassium hexacyanferrate (III) spray,
p-anisaldehydesulfuric acid spray, ethanolamine diphenylborate, chloranil reagent spray) to verify the presence of wide
varieties of chemical materials such as alkaloids, glycosides, steroids, flavonoids, saponins, tannins and terpenes making
use of standard methods (Touchstone JC et al., 1978; Jork H et al., 1994).

Determination of Total Phenolic Content (TPC)

Total phenolic content of BGF extract was determined by using Folinphenol reagent method described
by (Manian et al. 2008). A stock solution of plant extracts was prepared in different aliquots (0.2, 0.4, 0.6, 0.8, 1.0 mg/ml).
Two ml of plant extracts was taken in test tubes and l ml of Folinphenol reagent was added (FC reagent was dissolved in
distilled water with 1:1 ratio). Then 5 ml of 20% sodium carbonate was added in each tube and subsequently the
combination was blended competently with vortex mixer after which the experiment tubes were kept in the dark for 40
min. Absorbance spectra were recorded at 725 nm using glass cuvettes. To decrease commonplace error, the reaction was
carried out in triplicate and the results were expressed in milligrams of gallic acid equivalent (mg GAE).

Determination of Total Flavonoid Content (TFC) by Colorimetric Process

Complete flavonoid content material of flower extracts was determined by Manian et al. (2008). Concisely, 100 l
of each and every plant extract (1 mg/ml) was once dissolved in corresponding solvents after which extracts had been made
up to 1 ml using distilled water followed by the addition of 75l of 10% sodium nitrate solution. After 6 min interval, 150

Impact Factor (JCC): 4.8136 NAAS Rating: 4.13

Antioxidant and Antibacterial Activities of Methanol 329
Extract of Blepharis Glomerans Flowers

l of 5% aluminium chloride solution and 0.5 ml of 1 M NaOH in test tubes was added. The mixture samples have been
made as much as 2.5 ml by using distilled water. The spectrum absorbance values had been read at 510 nm. Results had
been expressed as mg/g butylated hydroxytoluene (BHT) equivalents.

Antioxidant Evaluation
DPPH Assay

Free radical scavenging activity was determined by using the stable radical DPPH making use of the process of
Siddhuraju and Becker (2003). Crude extract samples were set at 100 g/ml, the plant extract of 2 ml was taken in test
tubes and 3ml of 0.3 mM methanolic solution of DPPH was added, mixed well and allowed to incubate at 300C for 20 min.
The absorbance value of the sample was measured at 517 nm. The percentage of DPPH radical scavenging endeavor was
calculated by using the following formula

% DPPH radical scavenging activity = [ x100]

ABTS Assay

The 2,2-azino-bis-three-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay is used for the determination of

whole antioxidant activity of a plant extract. Inactive form of ABTS+ free radical cation is transformed to the lively form
by means of reacting 7 mM ABTS aqueous solution with 2.4 mM potassium persulphate in the dark for 1216 h at room
temperature. The solution was diluted with methanol (1:89 v/v) and equilibtated at 300 C. Afterwards, 1 ml of ABTS
solution was added with 100 l of plant extract, and after an initial mixing of 30 min the absorbance value was measured at
734 nm. The percent of scavenging ABTS activity was calculated by using the following formula (Manian et al., 2008).

( )
% ABTS radical cation activity= [ 100]

Agar Disc Diffusion Method using B. Glomerans Flower Extracts

The methanol extract of BGF extract was checked for its potential biomedical application of antibacterial activity
using the agar disc diffusion method. Gram positive and gram negative human pathogenic bacteria were used for the
experiment and cultures were purchased from MTCC. Bacterial cultures of Escherichia coli (MTCC 1560), Staphylococcus
aureus (MTCC 9542 ), Bacillus subtilis (MTCC 3055), Salmonella typhimuriun (MTCC 3224), Klebsiella pneumoniae
(MTCC 2403), Enterobacter aeruginosa (MTCC 7661), Pseudomonas fluorescens (MTCC 2268) were used. Authentic
bacterial cultures have been sub cultured in nutrient broth. The MHA nutrient agar was prepared with distilled water.
The medium was autoclaved at 1210C and at a pressure of 15 lbs for 20 min. The sterile medium was poured into
autoclaved Petri plates and allowed to dry for a couple of minutes. Then, the aesthetic bacteria had been swabbed on agar
plates. The surface of the agar medium is placed on a ready standard disc (streptomycin 50 g/ml) as control. The stock
flower extract was prepared in 100 mg/ml. After that, the crude flower extracts were prepared at various concentrations of
50 l, 100 l, 150 l, and 200 l and discs were soaked in the extracts overnight. After that, the disc was positioned in
bacterial swabbed agar plates and incubated for 24 hours at 370 C. Subsequently, an incubation zone of inhibition was
examined with the naked eye. Results have been found and the zone of inhibition (ZOI) was measured by using zone
reader. The experiments were repeated thrice for better reproducibility.

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330 Siva Kesavarao Kommula, Prasad Undrajavarapu & Krishna Sowjanya Kanchana

Minimal Inhibitory Concentration (MIC)

MIC was determined by way of the microdilution process of Hammer et al. (1999) with minor changes. MIC is
outlined as the lowest attention of drug which controls microbial population growth. In this work, we prepared different
concentrations of methanol plant extracts (100, 50, 25, 12.5 mg/ml) to find out the strong concentration for inhibition of
bacterial growth. The MIC assay was done using 96 well plate, filled with 50 l of nutrient agar broth and 30 l of
bacterial culture, after which for treatment 30 l of plant extract was added. Plates were incubated at 370 C for 24 h.
Incubated plates were read at 560 nm in a microplate reader and values are tabulated.

STATISTICAL ANALYSIS

By using SPSS 19.0 software all statistical analyses were performed. All the quantitative data were presented as
mean standard deviation (SD). Differences among means were analyzed by one-way ANOVA test, followed by Tukey
HSD and student test. Statistical significance was set at p<0.05.

RESULTS
Identification of Secondary Metabolites, Phenolics and Flavonoids in B. Glomerans Flowers

Crude extract shown positive results of alkaloids, cardiac glycosides, steroids, saponins, flavonoids, terpenoids
and phenolic compounds. Furthermore, methanol extracts of BGF phenolic and flavonoid contents of 51.4 mg GAE/g and
38.9 mg/g respectively.

Free Radical Scavenging Activity on B. Glomerans Flowers

DPPH and ABTS radical scavenging activity of methanol extract was shown in the below table.

Table 1: Antioxidants Activity of B. glomerans Flower Extract

Antioxidant Assay Standard Methanol
DPPH assaya,b 73.14 0.07* 47.70 0.07
ABTS assaya,b 81.31 0.05* 44.80 0.04

*Ascorbic acid.a Mean value (n = 6) with significant difference at P < 0.05. b

Percentage of inhibition due to
extract concentration of 100 g/ml.

Mehanolic extract of BGF shown activity (47.7%) compared with the galic acid standard. ABTS free radical
scavenging activity was analyzed using BHT as standard. Mehanolic extract of B. glomerulans showed activity (44.8%)
compared with standard.

Antibacterial Activity of B. glomerulans

Methanolic extract of BGF shown higher inhibitor activity against K.. pneumoniae fallowed by E.coli, B.subtilis,
entero bacter aeroges, pseudomans fluorescens.The least activity was measured in S.typhi and S. aureus. MIC range was
calculated to be 25.0 mg/ml as the lowest concentration of methanol flower extract against K. pneumoniae.

Impact Factor (JCC): 4.8136 NAAS Rating: 4.13

Antioxidant and Antibacterial Activities of Methanol 331
Extract of Blepharis Glomerans Flowers

Figure 1: Antimicrobial Activity of Methanol Extract of B. Glomerulans

DISCUSSIONS

Several biochemical processes influence the overproduction of reactive oxygen species. Among all biochemical
processes oxidative stress plays key role in the generation of free radicals. Elevated generation Of free radical formation
brought on cell damage and prompted many dysfunctions like atherosclerosis, myocardial infarction, melanoma and
neurogenerative disorders in human beings. But, common antioxidant compounds are helpful in repairing free radical
formation in cells and properly manage various chronic disorders (Fakruddin et al., 2012). Antioxidant potential is based
on the composition of exclusive phenolic contents present in the Scutellaria litwinowii extracts (Bazzaz et al., 2011).
Antioxidant tests are incredibly targeted and touchy to temperature and incubation interval. Physiochemical properties of
the sample are very crucial for analysing antioxidant potentiality.

Similarly, our results expressed methanol extract had moderate antioxidant activity observed using DPPH and
ABTS methods in this concept, traditional antioxidants obtained a prominence as they're on the whole free from side
effects, much less expensive and considerable in many plant sources (Cai Y and Luo Q, 2003). Tremendous quantity of
medicinal plants were investigated for their antioxidant properties. Common antioxidants either within the type of raw
extracts or their chemical components are very powerful to avoid the damaging tactics caused by using oxidative stress
(Akinmoladun A.C et al., 2008; zen T, et al., 2010). Recently, it has come to be evident that phenolic common products
may diminish oxidative stress by way of oblique antioxidant action (Khatoon M et al., 2013; Islam S et al., 2003; Anjaneya
S.R et al., 2012). Polyphenols were discovered in BGF. Plant polyphenols are synthesized from phenylalanine or from its
precursor shikmic acid. These phenolics are major dietary antioxidants for the reason that they have got the ideal structural
chemistry for free radical scavenging activities, and were proven to be extra powerful antioxidants in vitro than vitamins
E and C on a molar basis (Ribeiro S.M.R et al., 2008). Polyphenols exhibit huge range of organic effects corresponding to
safety of LDL oxidation in vivo with significant consequences in atherosclerosis and likewise guard DNA from oxidative
damage with essential consequences in the age-associated progress of some cancers (Nidyaletchmy S.R et al., 2012).
Our findings suggested that BGF rich in phenolic and flavonoid contents which are the major contributor to scavenge the
free radicals in oxidation pathways.

The outcome acquired from correlation between polyphenols (phenol and flavonoid) and DPPH scavenging
advised that phenolic compounds are the dominant contributors to the antioxidant properties of the extract/fractions.
Additionally it is stated that secondary metabolites in the extracts corresponding to polyphenols, phenolic acids,
flavonoids, diterpenes, tannins, phytosterols, fatty acid esters, phenylpropanoids, alkaloids and glycosides are major

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332 Siva Kesavarao Kommula, Prasad Undrajavarapu & Krishna Sowjanya Kanchana

bioactive compounds which have primary significance in medicinal chemistry (Maestri D.M et al., 2006). Hence, the
antioxidant property may also come from different antioxidant presence in the fractioned extract as well.

Recently, the growing resistance rates of bacterial strains and manage of the progress of pathogens are huge
challenge. Developing extra powerful antibacterial compounds using plant extracts is foremost in inhibiting bacterial
growth rate. Clinically challenging S. Aureus strains are a most important reason of community and health related
infections with an estimated mortality rate of around 710% (Tamokou et al, 2012). Methanol extract of BGF exhibited
higher activity in K. pneumoniae, E.coli and B.subtilis. Plants contain bio flavonoids and phenolics, which are marker
compounds for antimicrobial and antioxidant activities.

CONCLUSIONS

Crude methanol extract of B. glomerans showed antioxidant and antibacterial effect, it can be assumed that
different active secondary metabolites were present in this extract, but the exact mechanisms still require further research.

CONFLICT OF INTEREST

We have declared that there is no conflict of interests in this study.

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